The long term objective of this proposal is to understand the cellular mechanisms of the opioid-like peptide orphanin FQ (OFQ) in the anterior pituitary corticotroph tumor cell line AtT-20. My specific aims are: (1) To demonstrate that OFQ can interact specifically at the ORL-1 (Opioid Receptor-like 1) to stimulate release of ACTH from AtT-20 cells. This will be accomplished by measuring ACTH secretion from AtT-20 cells in response to OFQ. ORL-1 mRNA will be measured and binding studies conducted to confirm the presence of ORL-1 in AtT-20 cells. There is no known antagonist for ORL-1, thus antisense will be used to determine if OFQ is acting specifically through ORL-1. (2) To demonstrate that OFQ-stimulated ACTH secretion occurs via a G-protein coupled mechanism which involves an increase in inositol phosphate (IP) levels and (Ca2+)i. This will be accomplished by measuring IP stimulation from AtT-20 cells in response to OFQ. The effects of GTP-gammaS and GDP-betaS on OFQ-stimulated IP secretion will be examined, as well as the effects of pertussis toxin. In addition, the effects of Ca2+ channel blockers including nifedipine and omega-conotoxin on IP and ACTH production will be determined. The role of internal Ca2+ stores in OFQ-stimulated ACTH secretion will also be examined by treating cells with thapsagargin or ryanodine. OFQ has not previously been shown to increase levels of IPs in any cell system, nor has the role of OFQ in neuroendocrine secretion been examined. An understanding of the regulatory role of OFQ in neuroendocrine secretion may be potentially very useful in the treatment of hormonal disorders, or, conversely, OFQ may produce adverse effects on neuroendocrine function. The studies proposed here will greatly enhance our understanding of the role of OFQ in neuroendocrine secretion and expand our knowledge of the signalling systems employed by this peptide.